Limits...
Discoidin domain receptors regulate the migration of primary human lung fibroblasts through collagen matrices.

Ruiz PA, Jarai G - Fibrogenesis Tissue Repair (2012)

Bottom Line: Transwell migration experiments showed that normal human lung fibroblast (NHLF) transmigration through collagen I-coated inserts is mediated by DDR2 and the DDR2-associated signaling kinases JAK2 and ERK1/2, but not DDR1.Our results suggest a mechanism by which the presence of collagen I in situations of excessive matrix deposition could induce fibroblast migration through basement membranes through DDR2 activation and subsequent DDR1 and MMP-2 gene expression.This work provides new insights into the role of DDRs in fibroblast function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Novartis Institutes for Biomedical Research, Respiratory Disease Area, Wimblehurst Road, Horsham, RH12 5AB, UK. gabor.jarai@novartis.com.

ABSTRACT

Background: The two discoidin domain receptors (DDRs), DDR1 and DDR2 are receptor tyrosine kinases (RTKs) with the unique ability among RTKs to respond to collagen. We have previously shown that collagen I induces DDR1 and matrix metalloproteinase (MMP)-10 expression through DDR2 activation and a Janus kinase (JAK)2 and extracellular signal-regulated kinase (ERK)1/2-mediated mechanism in primary human lung fibroblasts suggesting that these signaling pathways play a role in fibroblast function. Fibroblasts can traverse basement membrane barriers during development, wound healing and pathological conditions such as cancer and fibrosis by activating tissue-invasive programs, the identity of which remain largely undefined. In the present work, we investigated the role of DDRs and DDR-associated signal transduction in these processes.

Results: Transwell migration experiments showed that normal human lung fibroblast (NHLF) transmigration through collagen I-coated inserts is mediated by DDR2 and the DDR2-associated signaling kinases JAK2 and ERK1/2, but not DDR1. Additionally, experiments with specific small interfering (si)RNAs revealed that collagen I-induced expression of MMP-10 and MMP-2 is DDR2 but not DDR1 dependent in NHLFs. Our data showed that collagen I increases NHLF migration through collagen IV, the main component of basement membranes. Furthermore, basal and collagen I-induced NHLF migration through collagen IV-coated inserts was both DDR2 and DDR1 dependent. Finally, DDR2, but not DDR1 was shown to be involved in fibroblast proliferation.

Conclusions: Our results suggest a mechanism by which the presence of collagen I in situations of excessive matrix deposition could induce fibroblast migration through basement membranes through DDR2 activation and subsequent DDR1 and MMP-2 gene expression. This work provides new insights into the role of DDRs in fibroblast function.

No MeSH data available.


Related in: MedlinePlus

Collagen I induces normal human lung fibroblast (NHLF) migration through collagen IV. NHLFs were grown on non-coated 8.0 μm polycarbonate inserts, or inserts coated with collagen IV (10 μg/cm2) or fibronectin (5 μg/cm2). Cells were serum-starved for 24 h and incubated with collagen I (25 μg/mL) or vehicle (acetic acid, 0.1 M) for 24 h. The bottom chamber was treated with cell dissociation buffer and acetomethoxycalcein (calcein AM) for 1 h at 37°C. Fluorescence of solution with detached cells was measured at 485 nm excitation and 520 nm emission. Results are representative of mean fold increase ± SD of two independent experiments performed in triplicate (n = 6, **P < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3298810&req=5

Figure 1: Collagen I induces normal human lung fibroblast (NHLF) migration through collagen IV. NHLFs were grown on non-coated 8.0 μm polycarbonate inserts, or inserts coated with collagen IV (10 μg/cm2) or fibronectin (5 μg/cm2). Cells were serum-starved for 24 h and incubated with collagen I (25 μg/mL) or vehicle (acetic acid, 0.1 M) for 24 h. The bottom chamber was treated with cell dissociation buffer and acetomethoxycalcein (calcein AM) for 1 h at 37°C. Fluorescence of solution with detached cells was measured at 485 nm excitation and 520 nm emission. Results are representative of mean fold increase ± SD of two independent experiments performed in triplicate (n = 6, **P < 0.01).

Mentions: There is a large body of evidence highlighting the impact of various ECM proteins on cellular function including cell differentiation, attachment, and migration. To address the question whether collagen I is able to induce NHLF migration we performed transwell migration assays using inserts coated with different ECM proteins including network-forming collagen IV and fibronectin, as well as non-coated inserts. Our results show that the level of constitutive NHLF migration was higher through inserts coated with fibronectin compared to non-coated, and collagen IV-coated inserts, and was not further enhanced upon collagen I stimulation. In contrast, collagen I significantly enhanced constitutive NHLF transmigration through both non-coated and collagen IV-coated inserts (Figure 1). These results indicate that different ECM components have different effects on fibroblast migration and suggest a possible link between collagen I-induced gene expression and fibroblast migration through basement membranes.


Discoidin domain receptors regulate the migration of primary human lung fibroblasts through collagen matrices.

Ruiz PA, Jarai G - Fibrogenesis Tissue Repair (2012)

Collagen I induces normal human lung fibroblast (NHLF) migration through collagen IV. NHLFs were grown on non-coated 8.0 μm polycarbonate inserts, or inserts coated with collagen IV (10 μg/cm2) or fibronectin (5 μg/cm2). Cells were serum-starved for 24 h and incubated with collagen I (25 μg/mL) or vehicle (acetic acid, 0.1 M) for 24 h. The bottom chamber was treated with cell dissociation buffer and acetomethoxycalcein (calcein AM) for 1 h at 37°C. Fluorescence of solution with detached cells was measured at 485 nm excitation and 520 nm emission. Results are representative of mean fold increase ± SD of two independent experiments performed in triplicate (n = 6, **P < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298810&req=5

Figure 1: Collagen I induces normal human lung fibroblast (NHLF) migration through collagen IV. NHLFs were grown on non-coated 8.0 μm polycarbonate inserts, or inserts coated with collagen IV (10 μg/cm2) or fibronectin (5 μg/cm2). Cells were serum-starved for 24 h and incubated with collagen I (25 μg/mL) or vehicle (acetic acid, 0.1 M) for 24 h. The bottom chamber was treated with cell dissociation buffer and acetomethoxycalcein (calcein AM) for 1 h at 37°C. Fluorescence of solution with detached cells was measured at 485 nm excitation and 520 nm emission. Results are representative of mean fold increase ± SD of two independent experiments performed in triplicate (n = 6, **P < 0.01).
Mentions: There is a large body of evidence highlighting the impact of various ECM proteins on cellular function including cell differentiation, attachment, and migration. To address the question whether collagen I is able to induce NHLF migration we performed transwell migration assays using inserts coated with different ECM proteins including network-forming collagen IV and fibronectin, as well as non-coated inserts. Our results show that the level of constitutive NHLF migration was higher through inserts coated with fibronectin compared to non-coated, and collagen IV-coated inserts, and was not further enhanced upon collagen I stimulation. In contrast, collagen I significantly enhanced constitutive NHLF transmigration through both non-coated and collagen IV-coated inserts (Figure 1). These results indicate that different ECM components have different effects on fibroblast migration and suggest a possible link between collagen I-induced gene expression and fibroblast migration through basement membranes.

Bottom Line: Transwell migration experiments showed that normal human lung fibroblast (NHLF) transmigration through collagen I-coated inserts is mediated by DDR2 and the DDR2-associated signaling kinases JAK2 and ERK1/2, but not DDR1.Our results suggest a mechanism by which the presence of collagen I in situations of excessive matrix deposition could induce fibroblast migration through basement membranes through DDR2 activation and subsequent DDR1 and MMP-2 gene expression.This work provides new insights into the role of DDRs in fibroblast function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Novartis Institutes for Biomedical Research, Respiratory Disease Area, Wimblehurst Road, Horsham, RH12 5AB, UK. gabor.jarai@novartis.com.

ABSTRACT

Background: The two discoidin domain receptors (DDRs), DDR1 and DDR2 are receptor tyrosine kinases (RTKs) with the unique ability among RTKs to respond to collagen. We have previously shown that collagen I induces DDR1 and matrix metalloproteinase (MMP)-10 expression through DDR2 activation and a Janus kinase (JAK)2 and extracellular signal-regulated kinase (ERK)1/2-mediated mechanism in primary human lung fibroblasts suggesting that these signaling pathways play a role in fibroblast function. Fibroblasts can traverse basement membrane barriers during development, wound healing and pathological conditions such as cancer and fibrosis by activating tissue-invasive programs, the identity of which remain largely undefined. In the present work, we investigated the role of DDRs and DDR-associated signal transduction in these processes.

Results: Transwell migration experiments showed that normal human lung fibroblast (NHLF) transmigration through collagen I-coated inserts is mediated by DDR2 and the DDR2-associated signaling kinases JAK2 and ERK1/2, but not DDR1. Additionally, experiments with specific small interfering (si)RNAs revealed that collagen I-induced expression of MMP-10 and MMP-2 is DDR2 but not DDR1 dependent in NHLFs. Our data showed that collagen I increases NHLF migration through collagen IV, the main component of basement membranes. Furthermore, basal and collagen I-induced NHLF migration through collagen IV-coated inserts was both DDR2 and DDR1 dependent. Finally, DDR2, but not DDR1 was shown to be involved in fibroblast proliferation.

Conclusions: Our results suggest a mechanism by which the presence of collagen I in situations of excessive matrix deposition could induce fibroblast migration through basement membranes through DDR2 activation and subsequent DDR1 and MMP-2 gene expression. This work provides new insights into the role of DDRs in fibroblast function.

No MeSH data available.


Related in: MedlinePlus